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FILE - In this Tuesday, Aug. 27, 2013 file photo, Russian air defense system missile system Antey 2500, or S-300 VM, is on display at the opening of the MAKS Air Show in Zhukovsky outside Moscow, Russia. The Russian military ... more >

Russian military leaders are threatening to call off the longstanding agreement with the U.S. to share the skies above Syria, potentially putting American aircraft in the region at risk.

On Thursday, Pentagon press secretary Peter Cook pressed Moscow to adhere to the deal, saying it was vital in order to “avoid misunderstanding and miscalculation” between the two countries.

The dispute adds yet another complication to the effort to end the Syrian civil war, where Russia and the U.S. are backing different sides, and take the fight to Islamic State and other jihadi groups that have set up a base in Syria amid the six years of fighting.

“Up to this point we believe served its purpose in avoiding misunderstanding and miscalculation [and] avoiding potential problems in the air over Syria,” Mr. Cook said of the pact, reached between Washington and Russia last October.

The deal, which has prevented collisions between Russian fighters and U.S. aircraft conducting airstrikes against the Islamic State in Syria, remains “an effective means of communicating with the Russians at a time when we continue to have very significant difference with the Russians and their activities” in the country, he added during a briefing at the Pentagon.

A second cease-fire brokered by Moscow and Washington broke down last month, with both sides blaming the other for the breakdown. Mr. Cook’s warning Thursday came just days after Russian forces deployed several anti-aircraft missile systems to Syria. The S-300 midrange and S-400 long-range missile systems were sent to defend Russia’s main airbase in Syria near the port city of Latakia, while bolstering the regime of Syrian President Bashar Assad, a longtime ally of Moscow.

On Thursday, Russia announced that its troops manning the new missile systems would be unwilling or unable to adhere to the airspace pact, amid ramped-up Russian airstrikes against rebel forces fighting to overthrow Mr. Assad.

“It must be understood that Russian air defense missile crews will unlikely have time to clarify via the hotline the exact flight program of the missiles or the ownership of their carriers,” Russian Defense Ministry spokesman Maj. Gen. Igor Konashenkov said in a statement Thursday.

Mr. Cook declined to characterize the deployment of the missile systems as a direct threat to U.S. aircraft operating in the country. However, he noted the rebel fighters battling Mr. Assad’s forces do not have or use attack aircraft.

The missile batteries were sent to the Russian air base in Latakia days after a U.S. airstrike hit a group of Syrian troops near the eastern province of Deir al-Zour. Russian defense officials claim the airstrikes killed over 60 government troops, wounding nearly 100.

U.S. commanders believed they were attacking Islamic State fighters moving through the region. American fighters only stopped firing after Russian military advisers reached out to U.S. commanders in the region, notifying them of their mistake.

Mr. Cook on Thursday said the command investigation into the mistaken airstrike, being led by U.S. Central Command, is still underway. The Pentagon would provide the findings to its Russian counterparts, if Moscow makes such a request, but had no plans to involve Russia in the investigation, he added.

Russian and Syrian warplanes have unleashed thousands of airstrikes on the rebel stronghold of Aleppo in the weeks since the collapse of a cease-fire deal brokered by Secretary of State John F. Kerry and Russian Foreign Minister Sergei Lavrov in late September.

Russia’s entry into the war last year, along with Moscow’s use of so-called “bunker-buster” munitions and cluster bombs — the latter of which are banned under the international rules of war — have only upped the ante in Syria.

The recent attacks are prompting calls for the U.S. and its allies to establish a no-fly zone over the besieged city, to prevent the imminent slaughter of the city’s roughly 275,000 residents.

In an interview with Danish television, Mr. Assad on Thursday denied that his government is targeting hospitals and civilian infrastructure as it presses the fight against rebel forces.

“To say that this is our aim as a government, [that] we give the orders to destroy hospitals or schools or to kill civilians, this is against our interest,” Mr. Assad.